1. Field of the Invention
The present invention relates essentially to a process of producing a dehydrated polysaccharide gel containing microorganisms for preparing fermented drinks, the dehydrated gels obtained therefrom, and their use for the preparation of fermented drinks, notably for the second fermentation of wine to produce sparkling wine and notably champagne.
2. Description of the Related Art
In the state of the prior art, it is known that immobilized microorganisms can be used in the production of fermented drinks such as wine and beer (FR-A-2 320 349 and FR-A-2 359 202). Their use has also been suggested for classical champagnization (FR-A-2 432 045) as well as for the manufacture of sparkling drinks with a variable degree of alcohol (FR-A-2 601 687). These publications, as well as others (in particular JP-A-57-150 385 and EP-A-173 915) have emphasized the performances of reactors with immobilized cells.
These techniques have made it possible to carry out fermentations with mixtures of microorganisms of different categories (mixture of lactic acid bacteria and mixture of yeast).
However, the implementation of the process on an industrial scale came up against the difficulty of having particles available which are capable of being preserved for a long period.
Microorganisms in the immobilized form, for instance in a gel, may be used without an appreciable drop of activity over long periods when the nutrition of the microorganisms concerned is respected.
It has been possible to note a toxicity of the products of fermentation which leads to a partial ageing of the cells (article by DIVIES et al. in Annales de microbiologie, 1977, pages 349-358).
It was thus preferred in this particular case to use a battery of reactors of defined life time and to carry out the partial renewal of the microbial particles in a programmed manner.
Problems of mechanical resistance of the gel entrapping microorganisms have also arisen and are described in JP-A-57-150 385.
It thus appears crucial industrially to plan the elaboration of the particles for inclusion of the microorganisms and to centralize the production of them. It is also necessary to bring about the inclusion of the microorganisms in the gels which ensures an excellent viability of them over a period of time.
However, it because apparent that the microorganisms entrapped in the gels were rather sensitive and could not survive after a storage over a long period of time.
In order to overcome this drawback, it has already been suggested to store the gel in a dry form.
The commercial preparations of microorganisms entrapped with a dried gel naturally need to be capable of rehydration and to maintain excellent viability after rehydration, and stability of the structure of the gel as well as its mechanical properties.
Certain solutions have been proposed which permit storage at a relatively low temperature range of about 4 to 10.degree. C. in a protective packaging over periods of time which may reach 6 months to 1 year (see BEKER and RAPOPORT in Advances in Biochemical Engineering and Biotechnology, Volume 35, 1987, pages 128 to 171). In the paragraph heading "Applications of dehydration for production of active dry yeasts", beginning on page 162, and in particular the passage on page 163, lines 13 to 15, BEKER and RAPOPORT report that the usual amount of hydrophilic wetting agents used as protective drying agents is from 0.5 to 5% of the dry weight of the yeasts to be dried.
In addition, U.S. Pat. No. 3,407,072 states in column 2, lines 43 to 46, in relation to protective drying agents described in column 2, lines 8 to 11, concentrations ranging from about 2 to 40 g/kg of compressed yeasts to be dried. From the examples, it appears that this concentration is essentially the same when expressed per liter of yeasts in suspension. Therefore, this disclosure is consistent with the usual concentration of the protective drying agent set forth by BEKER and RAPOPORT hereinabove which ranges from 5 to 50 g/kg of material to be dried.
The TATE document EP-A-0 065 376 describes a process for the preparation of enzymes immobilized in a gel which is then dried if need be and, after drying, is put in contact with glycerol (page 10, 2nd paragraph and Claim 3). A placing in contact after drying does not make it possible to preserve the structure of the gel and renders its rehydration difficult. This problem is solved by the present invention and which will be described below.
The document FR-A-2 519 022 describes a process for the preparation of inocula with long viability and which have an improved resistance to temperature which comprises a drying of the microorganisms in accordance with various drying processes. In its introduction, this documents describes many prior art documents which relate to drying gels enclosing microorganisms.
According to this FR-A-2 519 022 document, at the beginning of the process, a culture of microorganisms is grown in a standard culture medium for several days.
A gelable polymer, notably a polysaccharide, such as xanthane or alginate, may be added to this culture medium.
After gelation, which enables the inclusion of a microorganism in the culture medium, drying is undertaken in order to reach a water activity value in the inoculum lower than 0.5, this value being maintained during storage, see claim 1 in particular. Preferably, the water activity in the inoculum is maintained below 0.3 and preferably even below 0.1, see claim 2.
It is to be noted that, according to this document, no consideration is taken regarding the special problem of the rehydration of the gel so as to obtain a rehydrated gel having a structure approximately identical to that it had before its dehydration.
Now, experiments have shown that the dehydrated or dried particles obtained by the method described in this document, could not be properly rehydrated. At best, in the presence of culture medium, the rehydration remains limited at 20% humidity, the particles always remaining very small, dried up, very hard and un-graded with respect to size.
If a supplementation with hydrophilic substances such as carrageenan or carob seed grain is carried out, this causes the gel to become brittle without improving the rehydration of the gelled polymer, and this is particularly true in the case of the use of an alginate. At best, too low viabilities imcompatible with an industrial use may be obtained.
Hence, it appears necessary to dispose of easily re-hydratable gels which contain a maximum of viable microorganisms after rehydration, and which do so even after a long period of storage.
U.S. Pat. No. 5,389,532 discloses a first improvement enabling obtaining dried or essentially dehydrated gels, which are easily re-hydratable, contain a satisfactory viable microorganisms content after rehydration and which can be stored during a long period of time.
In this patent, it is disclosed a pre-treatment of the gels entrapping the microorganisms in a solution containing at least 500 g/l of hydrophilic substances, wherein the best compound is sucrose, which is preferably used at about 1,000 g/l, prior performing a drying of the gels.
Now, it has been discovered that a pre-treatment with such a high concentration of hydrophilic substances, in particular sucrose or sorbitol, provides a drawback of rendering the drying more difficult with a corresponding risk of damaging the microorganism cells entrapped in the gel and or requiring the preparation of such highly concentrated solutions, which are also relatively costly on an industrial scale.